The extracellular matrix is an important regulator of cell and tissue behaviour. Proteins of the extracellular matrix, such as fibronectin, vitronectin, collagens and laminin bind to a number of cell receptors that control many medically important biological phenomena, such as angiogenisis, cell migration, tissue repair, cancer cell differentiation, platelet aggregation and homing of immune system cells and neuronal processes to target sites. An important family of receptors that bind to the extracellular matrix are the integrins.
Fibronectins are large extracellular matrix glycoproteins (˜450 KDa) that have been implicated in integrin binding. They are composed of two nearly identical disulfide bonded subunits, each subunit consisting of three types of repeating homologous modules termed FN-I, FN-II and FN-III repeats. In addition, alternatively spliced modules, called EDA, EDB and IIICS, can also be present (Mohri, 1997; Pankov and Yamada, 2002). Single modules or groups of modules may contain binding sites for different molecules, including sulfated glycosaminoglycans, DNA, gelatin, heparin and fibrin (Mohri, 1997; Pankov and Yamada, 2002; Yamada, 1989). Furthermore, fibronectins contains binding sites for about half of the known cell surface integrin receptors (Johansson et al., 1997; Plow et al., 2000). In particular, the FN-III10 repeat contains an RGD site that can bind α3β1, α5 β1, αvβ3, αvβ5, αvβ6, α8β1 and αIIbβ3 integrins, while the FN-II9 repeat contains the so-called “synergy site” PHSRN (SEQ ID NO: 51) that cooperates with RGD in the binding of α5β1 and aIIbβ3 (Busk et al., 1992; Dedhar and Gray, 1990; Gardner and Hynes, 1985; Johansson et al., 1997; Pankov and Yamada, 2002; Plow et al., 1985; Pytela et al., 1985; Smith et al., 1990; Takada et al., 1987; Vogel et al., 1990; Weinacker et al., 1994). The FN-III14-IIICS region contains three sites (called CS1, CS5 and H1) characterised by the presence of LDV motif and accessory REDV (SEQ ID NO: 52) and IDA sites involved in α4β1, and α4β7 recognition (Johansson et al., 1997; Pankov and Yamada, 2002). Moreover, the FN-III5 repeat contains the KLDAPT (SEQ ID NO: 53) sequence that bind α4β1 and α4β7 (Moyano et al., 1997) whereas FN-I19 and FN-II1-2 contain sites, not yet identified, that can interact with α5β1 (Hocking et al., 1998).
Primary and tertiary structure analysis of human fibronectin, showed that this protein contains two GNGRG (SEQ ID NO: 4) loops, located in FN-I5 and FN-I7 modules, that are conserved in bovine, murine, rat, amphibian and fish (Di Matteo et al.). Two additional NGR sites, less conserved, are also present in human FN-II1 and FN-III9. Recent experimental work showed that peptides containing the NGR motif can inhibit α5β1 and αvβ1-mediated cell adhesion to fibronectin (Koivunen et al., 1993).
The interaction between cell surface anchored integrins and extracellular matrix components have been implicated in angiogenesis, an important process in neonatal growth and in the pathogenesis of a large variety of clinical diseases including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, macular degeneration by neovascularization of retina and the like conditions. It is known that αvβ3 integrin, the vitronectin receptor, plays a critical role in angiogenesis (Hynes, 2002). Compounds able to inhibit the interaction of this integrin with extracellular matrix proteins are known to inhibit angiogenesis and tumor growth (Brooks et al., 1994; Brooks et al., 1995; Friedlander et al., 1995; Friedlander et al., 1996; Hammes et al., 1996). However, a problem associated with these inhibitors is their cross-reactivity with many integrin species.
In the light of increasing awareness of the role of the extracellular matrix in biological processes and disease development, there remains a need for novel therapeutics that target this area. The present invention addresses this need.
The antitumoral activity of some cytokines is well known and described. Some cytokines have already been used therapeutically also in humans (Fiers et al., 1995). For example, such cytokines as interleukine-2 (IL-2) and interferon α (IFNα) have shown positive antitumoral activity in patients with different types of tumors, such as kidney metastatic carcinoma, hairy cell leukemia, Kaposi sarcoma, melanoma, multiple mieloma, and the like. Other cytokines like IFNβ, the Tumor Necrosis Factor (TNF) α, TNFβ, IL-1, 4, 6, 12, 15 and the Colony Stimulating Factors (CFSs) have shown a certain antitumoral activity on some types of tumors and therefore are the object of further studies.
In general, the therapeutic use of cytokines is strongly limited by their systemic toxicity. TNF, for example, was originally discovered for its capacity of inducing the hemorrhagic necrosis of some tumors Carswell et al., 1975), and for its in vitro cytotoxic effect on different tumoral lines Helson et al., 1975), but it subsequently proved to have strong pro-inflammatory activity, which can, in case of overproduction conditions, dangerously affect the human body (Tracey et al., 1993).
As the systemic toxicity is a fundamental problem with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies are now under evaluation, aimed at reducing the toxic effects of this class of biological effectors while keeping their therapeutic efficacy. For example, WO01/61017 discloses a conjugation product between TNF or IFNγ and a ligand of the CD13 receptor; WO03/093478 discloses a pharmaceutical composition comprising a conjugate of a cytokine and a tumor targeting moiety wherein the cytokine is present in an amount which does not induce a negative feedback mechanism; and WO03/092737 discloses conjugates of various cytokines and tumor targeting moieties. The present invention provides novel cytokine conjugates that have therapeutic potential.